Method and apparatus for magnetically separating and resuspending super-paramagnetic particles in a solution

ABSTRACT

Superparamagnetic particles are separated from a suspension thereof in a first fluid and re-suspended in the same or another fluid in a container by subjecting them to a first application of a magnetic field to draw the particles to a surface or zone of the container and subsequently re-suspending the particles in the fluid within the container by a second application of a magnetic field. Apparatus is disclosed including a container (1), electromagnets (2,3) for producing the first and second applications of magnetic fields, reagent receptacles (7), and a wash flask (8). A particle counting device may also provided and such apparatus may be used to count cells.

This invention relates to a method and apparatus for separatingsuperparamagnetic particles from a fluid suspension thereof andre-suspending them in the same or another fluid.

It has been proposed, e.g. in EP 106873, and U.S. Pat. No. 3,970,518, touse superparamagnetic particles for the immobilisation or isolation of awide range of substrates. These may include proteins, nucleic acids,viruses and cells. Such particles have the advantage that they can bereadily separated together with the immobilised substrate from asuspension thereof, for example a reaction medium, by application of amagnet to the wall of the vessel containing the suspension whereupon theparticles are drawn to the wall as a relatively compact aggregate. Thefluid is then readily removed from the vessel and replaced by a secondfluid, for example a wash solution or a second reagent whereupon themagnetic field is removed and the aggregated magnetic particlesre-suspended by relatively vigorous agitation. On the very small scale,the operator may effect such agitation by flicking the container withhis finger but it will be appreciated that this will not be appropriateon the larger scale or in automated systems. It has been suggested that,rather than remove the magnet before re-suspension of the particles itmight be possible to rotate the magnet around the container and therebyre-suspended the particles. However, it has been found that rotation ofthe magnet produces a rolling wave of particles; the particles stayingas a compact aggregate but rolling over one another and thus continuingto entrap contaminants and reagents.

There is thus a need for a reliable and readily automated method ofre-dispersing superparamagnetic particles after magnetic aggregation. Itshould be noted that superparamagnetic particles do not retainmagnetisation as would be the case with magnetic particles. Thus, whenthe initial magnetic field is removed, there are no magnetic forcesbetween the particles and the aggregate is held together by compaction.

We have found that by the first application of a magnetic field and,subsequently, a second application of a magnetic field,superparamagnetic particles may be aggregated and efficientlyre-suspended without physical agitation. This finding is surprising inview of the fact that rotation of a magnet to change the magnetic fieldinfluencing the particle aggregate failed to produce effectivere-suspension.

One aspect of our invention provides a method of separatingsuperparamagnetic particles from a suspension thereof in a first fluidand re-suspending said particles in the same or another fluid wherebysaid suspension in a container is subjected to a first application of amagnetic field to draw said particles to a surface or zone of saidcontainer and subsequently said particles are re-suspended in the sameor another fluid within said container by a second application of amagnetic field to draw the particles into said fluid.

The invention has the advantages that it leads to rapid and efficientre-suspension of the particles with little clumping and subjects theparticles to relatively low shear. The method may be readily repeatedseveral times so that the particles may be washed or treated with one ormore reagents. In general, the particles will move across the chamberi.e., in a direction towards a central interior space of the chamberthat is inwardly spaced from the peripheral interior surface of acontainer defining the chamber, at a speed related to the magnetic fieldand will thus comprise a moving suspension which contacts the fluid inthe chamber particularly efficiently.

It will be clear to a person skilled in the art that the first andsecond applications of magnetic fields may be produced by the samemagnet; the magnet being removed from close proximity to the containerso that its magnetic field has little or no effect on the particles, andthen being returned to the container at a different location such thatit provides the second application of a magnetic field for re-suspensionof the particles. Conversely, the magnet may, if an electromagnet, beenergised to effect aggregation, de-energised and then re-energised at adifferent position relative to the container either by moving the latteror the electromagnet.

However, it is desirable to avoid unnecessary moving parts, especiallyin automated systems, so we prefer to use two spaced electromagnets togenerate the first and second magnetic fields. Use of electromagnets hasthe additional advantage that the strengths of the magnetic fields canbe varied with respect to time while they are being applied. Thus, forexample, an initially strong second magnetic field may be applied tore-suspend the particles and the strength of the field can then bereduced with time to avoid unnecessarily firm aggregation on theopposite wall of the container. In the case of successive aggregationsand suspensions it will be appreciated that with two opposedelectromagnets these can be energised alternately to produce the desiredfirst and second applications of magnetic fields and with suitabletiming it may be possible to keep the particles suspended in the centreof the chamber.

The invention also provides, as a second aspect thereof, apparatus toseparate superparamagnetic particles from a suspension thereof in afirst fluid and re-suspending said particles in the same or anotherfluid, said apparatus comprising a container having a surface or zonetowards which said particles may be drawn by a first application of amagnetic field, and from which surface or zone said particles may bedrawn into re-suspension by a second application of a magnetic field andmeans for producing said first and second applications of magneticfields.

The container normally has an inlet port and an outlet port and isadvantageously made of transparent material or at least has atransparent "window" to permit illumination of the interior. If desired,means may be provided for projecting light into the container throughsuch a window. The means for producing the first and second applicationsof magnetic fields preferably comprise two, spaced electromagnets.

The apparatus can readily form part of a small, inexpensive portableanalyser which will be described below.

It is preferred that the superparamagnetic particles are alsomonodisperse beads. An example of how to produce monodisperse bead isgiven in EP 106873 (Sintef). The term "monodisperse" used herein isintended to encompass size dispersions having a diameter standarddeviation of less than 5%. Advantageously, the beads are in the sizerange 1 to 10 microns, in particular the size range 3 to 6 microns, e.g.about 4.5 microns.

In order that the superparamagnetic particles can be used for theimmobilisation or isolation of substrates, for example proteins, nucleicacids or cells, a suitable ligand is applied to the particles. Suchligands include, inter alia lectins, antibodies and single strandednucleic acids. The ligands may be adsorbed onto the surface of eachparticle although it is preferred that they are bound in some way, suchas by covalent linking to functionalised groups on the particles.

It is surprising that ligands carried by superparamagnetic monodisperseparticles in the size range mentioned above react virtually as rapidlyas if free in solution. By using monodisperse particles the reactionrate of ligand binding (or other reactions at or near the particlesurface) and other parameters are particularly uniform. By usingsuperparamagnetic particles one avoids magnetic aggregation or clumpingof the particles during reaction, thus again ensuring uniform and rapidreaction kinetics.

The invention will find particular utility in cell separation. Therelatively low shear produced on re-suspension of the particles isadvantageous when viable cells are sought. The lack of clumpingmentioned above means that unwanted matter, such as undesired cells,which may have become physically entrapped when the particles were drawnto the surface will be free in the re-suspension fluid and unlikely tobe entrapped during subsequent washing steps.

In one embodiment of the invention the apparatus is combined with aparticle counting device. This may comprise an array of charge coupleddevices (CCDs) and imaging software as described in our co-pendingInternational application PCT/EP 9002121 claiming priority from GB8927742.0, filed 7 Dec. 1989. For example, a combined cell separator andparticle counting device may be used to count cells of interest in adiverse population, e.g. a blood sample, and provide a valuable tool fordiagnosis of disease. Briefly, magnetic beads coated with antibodiesagainst the desired cell type are mixed with a sample and fed to aseparation chamber according to the invention. The beads become attachedto the desired cells which are then drawn to one side of the chamber bya first magnetic field produced by a first electromagnet. Unwantedsample is then flushed from the chamber and the beads bearing thedesired cells are re-suspended in a washing buffer by the action of asecond magnetic field produced by a second electromagnet. The washingstep is repeated and the nuclei of the cells are then stained with a dyesuch as acridine orange. The beads are then drawn to the surface of thevessel by one of the magnetic fields and surplus dye is flushed from thechamber. The beads with cells attached are re-suspended by the action ofthe other electromagnet in a buffer containing detergent which leads tocell rupture and release of the stained nuclei. It is preferred to usestained nuclei rather than cells since nuclei tend not to exhibit such awide size distribution as cells.

The stained nuclei are flushed from the separation chamber into areading chamber. The reading chamber is illuminated with UV light froman appropriate source and the fluorescent light emitted by the stainednuclei passes through magnifying optics to produce an image on a CCDchip which comprises an array of charge coupled devices. An emittencefilter is preferably present between the chamber and the CCD chip toselect only light of the desired wavelength. It is possible to use aplurality of dyes which may selectively bind certain nuclei types inpreference to others and use a plurality of suitable emittance filtersin rotation such that the different nuclei types in the sample may becounted.

The invention also provides, as a third aspect thereof, apparatus forcounting comprising apparatus according to the second aspect of theinvention in combination with particle counter having an optical cellthrough which is passed a fluid containing particles to be counted,means for illuminating the particles in the optical cell and opticalmeans for providing an image of the particles in an array of chargecoupled devices such that the area of the image of each particle at thesaid array is approximately the same as the area of at least a singlecharge coupled device.

It will of course be apparent to the skilled person that the opticalcell and separation chamber can be the same vessel. As mentioned above,at least a part of the separation chamber is preferably transparent andit may be preferred to count whole cells separated from the originalsample.

The term CCD array as used herein refers to an array of photosensitiveCCDs which may for example, be of the frame-field or interline transfertype and may produce the required signals by current or voltage sensing.Such arrays are normally provided as integral CCD chips for use, forexample, in solid state cameras and one commercially available CCD chipis that available from Phillips N. V.

It is desirable that the magnification is such that fluorescent lightfrom a single nucleus will impinge on at least one CCD. Assuming that(i) a nucleus is generally circular in plan view and (ii) each CCD issubstantially square then each nucleus is preferably magnified so thatit substantially covers at least the area of a single a CCD and maypartially cover the area of 2×2 or 3×3 array of CCDs. As the nucleibearing fluid passes through the reading chamber the CCD chip isinterrogated, preferably ten times so that a plurality of readings aretaken for analysis. A plurality of readings for each sample ispreferable since errors caused by overlap of the nuclei can be accountedfor and size distribution of the nuclei can be calculated.

The invention will now be described by way of a non-limiting examplewith reference to the drawings in which:

FIG. 1 shows schematically an embodiment of apparatus according to theinvention in conjunction with a plurality of conduits and receptacles;and

FIG. 2 shows schematically a particle counting arrangement which can becombined with the apparatus shown in FIG. 1.

FIG. 1 shows a separation chamber 1 located between two spacedelectromagnets 2,3. The separation chamber comprises an inlet port 4 andan outlet port 5. A common feed conduit 6 connects a plurality ofreceptacles 7-11 which are fed by a common pressure line 12 controlledby a pump, not shown, which maintains all the vessels at the samepressure. Outflow from each receptacle 7-11 is controlled by arespective pinch valve 13 (shown schematically as a arrow with anunderscore). The volumes drawn off from the receptacles 7-11 thus dependonly on the time of opening of a respective valve 13 and the rate offlow of the liquid (determined by the sizes of the connecting tubes andthe viscosity of the liquid). Control of the valves 13 is automated by acomputer as indicated below. All the tubing connecting the receptaclesmay be disposable to avoid contamination and/or clogging problems.

The outlet port 5 of the separation chamber 1 is connected to a readingchamber 14 shown diagrammatically in FIG. 2. To one side of the readingchamber 14 there is a UV light source 15 and above the chamber 14 aremagnifying optics (represented schematically by a convergent lens 16),an emittence filter 17 and a CCD chip 18.

A combination of the apparatus shown in FIGS. 1 and 2 can be used as acell counter. A cell bearing sample is mixed with superparamagneticbeads having a cell selective antibody coating thereon and placed inreceptacle 7. Opening the respective valve 13 allows pressure from line12 to force the sample and beads into the separation chamber 1 via theconduit 6. The chamber 1, which will have been prefilled with washingbuffer, has a volume of about 2 ml; 100 μl of the sample may betransferred to the chamber. Energisation of one of the electromagnetsfor example electromagnet 2, aggregates the magnetic particles andselected cells to a zone 19 on the wall of the vessel 1 and thesupernatant is then evacuated through outlet 5 using 3 ml of a washsolution (buffer) from receptacle 8. The magnetic particles and cellsare then re-suspended by brief energisation of the other electromagnet,e.g. electromagnet 3, to apply a second magnetic field at a further zone20 and if necessary the two electromagnets can be energised alternatelyto cause the particles to move within fresh washing buffer backwards andforwards across the chamber several times. The washing procedure isrepeated at least once to ensure that no unwanted cells are trapped bythe magnetic beads and attached cells. The magnetic particles and cellsmay then be aggregated by electromagnet 2 and the wash solution replacedby a staining solution, typically washing buffer containing acridineorange, from receptacle 9. After re-energisation of the electromagnetsto re-suspend the particles the staining reaction is allowed to stainthe cell nuclei. Magnetic re-aggregation may then be followed byreplacement of the staining reagent by 3 ml of a wash solutioncontaining a detergent taken from receptacle 10. Energisation of one ofthe electromagnets 2 or 3 causes the magnetic particles and cells tomove across the chamber 1 and contact with the detergent causes thecells to rupture, whereupon the nuclei are released into the buffer. Oneelectromagnet, is then energised to aggregate the magnetic particles andall residues attached thereto; the buffer containing the stained nucleiis then flushed through with further washing buffer into the readingcell 14 (shown in FIG. 2). Finally, the system may be cleaned out bydrawing cleaning fluid from receptacle 11 through the chamber 1, e.g. 5ml of a suitable washing buffer. The sample inlet may be back-washedwith 2 ml buffer by using suitable valve arrangements. It should benoted here that either electromagnet 2 or electromagnet 3 may be usedfor aggregation of the magnetic particles; the other electromagnet beingused re-suspend the particles.

The reading cell 14 is made from optical quality glass or plastics. Theshape of the cell is designed to fit the optical lens system 16 and theCCD chip 18. The lens system 16 has a magnification ratio that will makea normal particle (e.g. a nucleus) trigger 3×3=9 CCDs in the CCD chip.By choosing this ratio, the analyzer faciliates measurements of bothsmaller and larger particles than normal. The CCD 18 chip is chosen onthe basis of its CCD density, sensitivity and reaction time.

Since the fluorescent dye, e.g. acridine orange, has a definitewavelength for its emission, a filter 17 is placed between the lenssystem 16 and the CCD chip 18 to filter out light of other wavelengths.

The CCD chip 18 gives continuous information regarding what is happeningin its viewing field. This information is transmitted to the processingunit in the analyzer (not shown).

The analyzer includes a computer (not shown) which handles allcommunication with the operator and all data analysis. Communicationwith the operator is via a conventional touch sensitive panel, such as akeyboard, and an LCD screen (neither of which are shown). At thebeginning of the analysis the computer will tell the operator to checkcertain functions and how to insert the sample. The processor willcontrol the valve function and monitor the pressure in the fluid system.The energisation of the magnetic fields is controlled and the data fromthe CCD chip 18 is collected.

The information from the CCD chim may, for example, be sampled as 10"frame freeze pictures", because the information from the CCD chip willbe real time information from a moving fluid. The information from the10 pictures will be processed by commercially available imagingsoftware. This software is capable of identifying the number ofilluminated objects and the size distribution.

The data obtained from the 10 pictures is treated statistically and theresults for the test are calculated.

The results of the analysis are presented on the LCD screen in terms ofthe number of cells and a histogram of the size distribution althoughother presentations will be apparent to the skilled person. Sizedistribution, for example, will give the operator a chance to see if thesample contained so many abnormal cells that further study is necessary.

A printer, e.g. a thermal printer, may be provided to print out a hardcopy of the test results.

We claim:
 1. A method of separating superparamagnetic particles from asuspension thereof in a first fluid and re-suspending said particles inthe same or another fluid whereby said suspension in a container issubjected to a first application of a magnetic field to drawsubstantially all of said particles to a surface or zone of saidcontainer to form an aggregate, removing said first application of amagnetic field from said container, and subsequently re-suspending saidparticles in the same or another fluid within said container by a secondapplication of a magnetic field at a different location relative to thesaid surface or zone.
 2. A method of separating superparamagneticparticles as claimed in claim 1 wherein the first and secondapplications of a magnetic field are provided by electromagnet means. 3.A method of separating superparamagnetic particles from a suspensionthereof in a first fluid and re-suspending said particles in the same oranother fluid whereby said suspension in a container is subjected to afirst application of a magnetic field to draw substantially all of saidparticles to a surface or zone of said container to form an aggregateand subsequently said aggregated particles which are in said aggregateare re-suspended in the same or another fluid within said container by asecond application of a magnetic field at a different location relativeto the said surface or zone to draw the particles into saidfluid,wherein the first and second applications of a magnetic field areprovided by electromagnet means comprising two spaced electromagnets togenerate first and second magnetic fields.
 4. Apparatus to separatesuper-paramagnetic particles from a suspension thereof in a first fluidand re-suspend said particles in the same or another fluid, saidapparatus comprising:a device for creating a first application of amagnetic field, removing said first application of said magnetic field,then creating a second application of a magnetic field; a rod-freecontainer having a peripheral interior surface including a portiontowards which substantially all of said particles are drawn by saidfirst application of a magnetic field to form an aggregate, and fromsaid portion said particles which are in said aggregate are drawn tore-suspension in said same or another fluid by said second applicationof a magnetic field at a location relative to said portion differentfrom said first application of a magnetic field to move said particlesin a direction away from said portion and towards a central interiorspace of said container that is radially inwardly spaced from saidperipheral interior surface so as to efficiently contact said same oranother fluid; wherein said device applies said first application of amagnetic field with a first electromagnet at a first position relativeto said container and applies said second application of a magneticfield with a second electromagnet located at a second position relativeto said container; and wherein said first electromagnet and said secondelectromagnet are fixed in relation to said container.
 5. Apparatus toseparate superparamagnetic particles as claimed in claim 4 in which thecontainer has an inlet port and an outlet port.
 6. An apparatus asclaimed in claim 4, further comprising:a particle counter incommunication with said container to receive one of said first fluid andsaid another fluid, said particle counter having an optical cell throughwhich said one of said first fluid and said another fluid is passed;means for illuminating said particles in the optical cell; and opticalmeans for providing an image of said particles on an array of chargecoupled devices such that the area of the image of each of saidparticles at said array is approximately the same as an area of at leasta single charge coupled device.
 7. An apparatus as claimed in claim 4,wherein said means for illuminating said particles provides radiationapproximately perpendicular to a light path from said optical cell tosaid array of charged coupled devices.
 8. An apparatus as claimed inclaim 4, further comprising at least one transmittance filter disposedbetween said optical cell and said array of charged coupled devices. 9.An apparatus as claimed in claim 4, further comprising means forprocessing signals from ones of said individual charge coupled devicesto provide information concerning at least the number of said particlespassing through the optical cell.
 10. An apparatus as claimed in claim6, wherein said container comprises said optical cell with a portionthereof being transparent.
 11. An apparatus comprising:a containerincluding a peripheral interior surface; a suspension in a first fluidincluding superparamagnetic particles; and a device that applies a firstmagnetic field to said container to cause substantially all of saidparticles to be drawn to a portion of said peripheral interior surfacefrom said suspension in said first fluid to form an aggregate andapplies a second magnetic field to said container to cause saidparticles to be drawn out in a direction away from said portion andtowards a central interior space of said container that is radiallyinwardly spaced from said peripheral interior surface and bere-suspended in said suspension in said first fluid or a suspension inanother fluid; wherein said device applies said first application of amagnetic field with a first electromagnet at a first position relativeto said container and applies said second application of a magneticfield with a second electromagnet located at a second position relativeto said container; and wherein said first electromagnet and said secondelectromagnet are fixed in regulation to said container.
 12. Apparatusfor separating superparamagnetic particles as claimed in claim 11,wherein said container includes an inlet port and an outlet port.